Abstract

Angiogenesis plays a central role in the growth and metastasis of
cancers. Strategies aimed at interfering with tumor blood supply offer
promise for new cancer therapies. Vitaxin (an
anti-ανβ3 antibody) interferes with blood
vessel formation by inducing apoptosis in newly generated endothelial
cells. This Phase I study evaluates the safety and pharmacokinetics of
Vitaxin in humans with cancer. Eligible patients demonstrated
progressive tumors with stage IV disease and an Eastern Cooperative
Oncology Group performance status ≤2. Treatment consisted of six
weekly infusions of Vitaxin. Escalating doses from 0.1 and 4.0
mg/kg/week were evaluated based on the expectation that plasma levels
would bracket the effective in vitro concentration.
Escalation beyond 4 mg/kg/week was limited by drug availability.
Adverse events were assessed weekly. Pharmacokinetics were performed
weekly through week 9. Clinical response was assessed at week 9.

Of 17 patients treated, 14 were evaluable for response. Treatment was
well tolerated with little or no toxicity. The most common side effect
was infusion-related fever, which could be controlled with prophylactic
antipyretics. Doses ≥1 mg/kg/week produced plasma concentrations
sufficient to saturate the ανβ3 receptor
in vitro (25 μg/ml). Vitaxin demonstrated a half-life
in excess of 5 days at higher doses with no accumulation over 6 weeks
of therapy. One patient demonstrated a partial response, and seven
patients demonstrated stable disease. Three patients received Vitaxin
beyond the first cycle of therapy. Each of these patients demonstrated
disease stabilization that in one case lasted 22 months.

INTRODUCTION

Many disease states (cancer, psoriasis, rheumatoid arthritis, and
diabetic retinopathy) are mediated, in part, by a pathological
angiogenic response (1, 2, 3, 4)
. In cancer, the
progressive growth of solid tumors is strictly dependent on their
ability to stimulate formation of new blood vessels to supply tumor
cells with oxygen and essential nutrients. Under normal physiological
conditions, the formation of new blood vessels is tightly regulated and
most tumors persist in a relatively benign or dormant state
(5)
. However, with neovascularization comes tumor growth
and the ability to shed tumor cells into the circulation (2, 6)
. This can lead to metastases.

Inhibition of angiogenesis was first proposed as an anticancer strategy
by Folkman (7)
in 1971. Support for this comes from
the observation that microvessel density can serve as a prognostic
indicator in early-stage breast carcinoma (8)
. A similar
correlation between microvessel density and prognosis has been made for
patients with colon carcinoma, prostate carcinoma, and melanoma
(8)
.

In view of this, antiangiogenic therapy appears an attractive and
rational approach for the treatment of solid tumors. To date, a number
angiogenic inhibitors have been identified (8)
, some
showing promising antitumor effects (4, 9, 10)
. However,
their molecular targets remain unclear (11)
, and many have
been associated with undesirable side effects.

Another approach to antiangiogenic therapy is to inhibit the adhesive
interactions required by angiogenesis vascular endothelial cells. The
migration of endothelial cells is dependent on their adhesion to extra
cellular matrix proteins, such as vitronectin, through a variety of
cell adhesion receptors known as integrins. Recent evidence indicates
that integrin ανβ3
plays a role in this process (12)
. Studies have shown the
enhanced expression ofα
νβ3 on newly
developing blood vessels in human wound tissue, tumors, diabetic
retinopathy, macular degeneration, and rheumatoid arthritis. However,α
νβ3 is not generally
found on blood vessels in normal tissues. In fact, in various animal
models, antagonists ofα
νβ3, such as theα
νβ3 specific
antibody, LM609, have been shown to decrease angiogenesis and induce
tumor regression (3)
or improve arthritic disease
(13)
, and this was associated with the induction of
apoptosis within the angiogenic blood vessels (4, 13)
.

Vitaxin (Applied Molecular Evolution, San Diego, CA) is a
humanized version of the LM609 monoclonal antibody that functionally
blocks the ανβ3
integrin. This antibody has been shown to target angiogenic blood
vessels (11)
and cause suppression of tumor growth in
various animal models (9)
. Based on the selectivity ofα
νβ3 as a marker of
angiogenic blood vessels and the effects of
anti-ανβ3 in
reversing disease in animal models, clinical trials were initiated to
evaluate the safety and pharmacokinetics of Vitaxin in late stage
cancer patients. Tumor response was assessed as a secondary end point.

PATIENTS AND METHODS

Eligibility.

Adult patients (aged ≥18 years) with measurable late stage cancer
were eligible for this study. Patients were required to have
histologically proven, advanced (stage IV), incurable malignancies
refractory to standard therapy. Pathology was determined by the
treating oncologist and verified by central review. In an effort to
evaluate Vitaxin therapy in a variety of different tumor types, study
entry of patients with either breast, lung, or colon cancer was limited
to three evaluable patients each. Inclusion criteria required patients
to have a good
PS3
(Eastern Cooperative Oncology Group PS of ≤2) and adequate bone
marrow, renal, cardiac, thyroid, and hepatic function. Additional entry
criteria included a life expectancy of ≥3 months, a hemoglobin ≥8.0
mg/dl, a WBC count ≥2500/mm3
, an absolute
granulocyte count ≥1000/mm3
, a platelet count≥
75,000/mm3
, a serum creatinine less than or
equal to two times the upper limits of normal, a normal serum
thyrotropin, an aspartate aminotransferase less than or equal to three
times the upper limits of normal, a bilirubin less than or equal to two
times the upper limits of normal, and a partial thromboplastin time
within normal limits.

Patients were excluded if they had known brain tumors, active
opportunistic infection, serious nonmalignant disease, or were
HIV-positive. Given that the effects of Vitaxin on embryogenesis are
unknown, pregnant women were excluded. All patients of reproductive
potential were required to practice birth control during the course of
the study. Patients who underwent any major surgery, chemotherapy, or
radiotherapy within 4 weeks of study entrance were also excluded.
Concomitant treatment with other investigational drugs was not
permitted.

This study was approved by the Institutional Review Board of the
Sharp/Sidney Kimmel Cancer Center. All patients gave informed consent
before participation (Table 1)⇓
.

Study Design.

The study was an open label, single center, dose escalating Phase I
trial with sequential cohorts receiving increasing doses of Vitaxin
(Table 2)⇓
. Dose escalation followed standard Phase I criteria (<33% incidence
of dose-limiting toxicity was required in prior cohorts before opening
the next cohort for patient accrual).

Treatment.

All patients received 6 weekly doses of Vitaxin at increasing doses
(0.1–4 mg/kg). Given that dose limiting toxicity was not expected with
Vitaxin, the doses chosen were anticipated to bracket the in
vitro concentration of Vitaxin required to saturate theα
νβ3 receptor and to
block endothelial cell migration. No intrapatient dose escalation was
allowed. Vitaxin was administered i.v. through a peripheral line as a
90-min infusion. Premedication was not given initially; however, with
the appearance of fever in a subset of patients shortly after
treatment, oral premedication with acetaminophen (650 mg) and
diphenhydramine (50 mg) was given before each infusion. Tumor restaging
was performed at week 9. Those patients demonstrating at least stable
disease were considered for additional cycles of treatment (weekly for
6 weeks) repeated every 10 weeks. Tumor biopsies were performed before
and after therapy to assess integrinα
νβ3 expression and
antibody binding.

Toxicity Assessments.

Patients were evaluated weekly during treatment with a physical exam
and blood work. Parameters followed throughout the study included serum
chemistries, thyroid function, coagulation tests, and anti-Vitaxin
antibodies. All toxicities were graded according to the National Cancer
Institute Common Toxicity Grading System. Patients were assessed at
week 12 for delayed toxicity.

Anti-Vitaxin Antibody Response.

Samples were taken for determination of human antimouse antibody titers
at baseline, and weeks 3, 6, and 9 after the start of treatment with
Vitaxin. Patient samples were run in comparison to two murine antihuman
antibodies. The first was a mouse antihuman κ. The second control was
pooled sera from two mice inoculated with Vitaxin. Assays were run in
plates coated with 200, 400, or 800 ng/ml of Vitaxin. Replicates were
run in triplicate at 1:20, 1:40, 1:80, and 1:160 dilutions against each
coating concentration.

Pharmacokinetics.

Plasma Vitaxin levels were measured before and at 0.08, 0.5, 1, 4,
8, 24, 48, and 72 h after completion of each Vitaxin infusion. In
addition, plasma Vitaxin levels were determined on day 1 of weeks 7, 8,
and 9. Patient samples were prepared for each time point and frozen
until the time of analysis.

Vitaxin levels were determined using an ELISA assay. This assay uses
the VnR, specificallyα
νβ3, as the
plate-bound ligand of Vitaxin. The VnR was purified from human
placentas. Dynatech Immulon 2 U-bottomed microtiter plates were coated
with 1.0 μg/ml VnR in Coating Buffer, sealed, and stored at
0°C-5°C from 1–21 days. After blocking and washing, the plates
were incubated with freshly thawed standards and controls, plus
appropriate dilutions of patient serum in blocking buffer with 5%
human serum. After a 1-h incubation and washing, the plates were
incubated for another hour with goat antihuman κ light chain
conjugated with horseradish peroxidase. The washed plates were then
loaded with ortho-phenylenediamine dihydrochloride substrate in
Citrate buffer for 20 min and stopped with sulfuric acid. Absorbance
was read at 490 nm and evaluated using SOFTmax PRO software. Standard
curves were required to have a correlation coefficient >0.985, and the
individual concentrations of standards were required to fall within
20% of their expected values. The absorbance values of the
three highest concentrations of sample dilutions that fell within the
range of the standards were selected in an Excel database, converted to
concentrations, and averaged.

Clinical Response Evaluation.

Evaluation of tumor burden was performed by comprehensive scans
(computed tomography, magnetic resonance imaging, X-rays) and
physical examination at baseline (study entry) and 3 weeks after
completion of therapy (week 9). Tumor responses were graded according
to standard procedures based on at least two indicator lesions. A
partial response was defined as less than a complete response but a>
50% decrease in the sum of the products of the cross sectional tumor
measurements of selected indicator lesions. Stable disease was defined
as a <50% decrease and a <25% increase in the sum of the products
of cross-sectional tumor measurements.

Statistical Considerations.

A patient was considered evaluable for response after receiving six
weekly doses of Vitaxin. Patients were considered evaluable for
toxicity after having received one dose of Vitaxin.

RESULTS

Patient Characteristics.

Seventeen adult patients (aged ≥18 years) were enrolled in this Phase
I study (February 1997 to February 1998). The characteristics of these
patients at study entry are presented in Table 1⇓
. The mean age was 61
years (range, 32–82) with eight females and nine males. Distribution
of PS was PS 0–24% (4 of 17), PS 1–59% (10 of 17), and PS 2–18%
(3 of 17). Pathological diagnoses included a range of tumor types, all
with stage IV disease. All patients had received prior therapy for
their cancer.

Vitaxin Toxicity.

Overall, Vitaxin therapy was well tolerated. No significant toxicity
was observed at any of the dose levels tested, and no patient was
required to stop treatment or have therapy delayed because of an
adverse event. Altogether, 88% (15 of 17) of patients experienced one
or more adverse events during the course of the trial, the majority of
which were grade 1 (68%) or 2 (32%). No toxicities greater than grade
2 were documented. The most frequent adverse events reported included
commonly seen antibody infusion reactions: fever, chills, nausea, and
flushing. Premedication was not given initially; however, several
patients demonstrated a fever lasting up to 2 h after treatment.
Oral premedication with acetaminophen and diphenhydramine before each
Vitaxin infusion appeared to prevent fever in subsequent patients.
Antibody infusion reactions decreased in incidence after the first
infusion. No clinically significant cardiac, renal, hepatic, or
hematological toxicity was observed.

Tumor Biopsy.

Most patients underwent a tumor biopsy during week 9. No appreciable
increase in bleeding or inhibition of wound healing was observed with
these biopsies. One patient with a leiomyosarcoma (patient 1) underwent
a scalp lesion biopsy during week 3 of treatment. This biopsy site did
not achieve complete hemostasis until a week later. The same patient
failed to demonstrate increased bleeding at the time of complete scalp
lesion resection (week 6), or at the time of a tumor biopsy of a
separate lesion (week 5).

Immunogenicity of Vitaxin.

Vitaxin antibodies were not detectable in any patient at week 9. Thus,
humanized Vitaxin does not appear to be immunogenic in any of the
patients treated to date.

Pharmacokinetics.

Vitaxin demonstrated a dose-dependent elimination half-life comparable
with the elimination half-life of other humanized antibodies in man
(Table 3⇓
; Ref. 14
). The 14.6-h half-life at the lowest dose (0.1
mg/kg/week) was 4- to 9-fold lower than the 63–138-h half-life seen at
higher doses (2–4 mg/kg/week). The volume of distribution for Vitaxin
is consistent with distribution throughout the plasma volume (0.05–0.1
liters/kg). The AUC for Vitaxin increased with increasing doses in a
manner that was not proportional to dose ranging from an AUC/dose of
412.3 μg·h/ml at 0.1 mg/kg/week to 2.1 mg·h/ml at 4 mg/kg/week.

Clinical Outcome.

After the first tumor evaluation at week 9, seven patients were shown
to have at least stable disease in their indicator lesions. One patient
(patient 1) with a widely disseminated leiomyosarcoma achieved a
partial response (45% of baseline) based on assessment of his
measurable disease (Fig. 1)⇓
. This same patient had a tumor nodule removed surgically from his
scalp during week 5, and had nonmeasurable metastatic disease to the
liver that was judged to be stable.

Time course of indicator tumor measurements for
patient 1 with a leiomyosarcoma. Sum of two measurable tumors. ▴,
start date of each 6-week cycle of Vitaxin.

Additional Cycles of Treatment.

Three patients received treatment beyond the initial 6-week cycle of
Vitaxin. Patient 1, with a leiomyosarcoma metastatic to the liver,
initially demonstrated edema and reddening at the site of a cutaneous
lesion on his scalp during week 3. This lesion (∼1 × 1 cm in
size) was removed and repaired with a small skin graft (∼3 × 3
cm) from the patient’s thigh. The patient’s remaining measurable
disease decreased in volume to 45% of baseline before his second cycle
of treatment initiated on week 17. At week 29, a small cutaneous lesion
appeared at the site of the prior scalp lesion. This lesion was limited
to the scar surrounding the prior skin graft and was treated with local
irradiation therapy resulting in complete resolution. Treatment with
Vitaxin continued until week 93 based on the impression that Vitaxin
was controlling a subset of the patient’s metastatic disease. At week
55, his dose of Vitaxin was increased 3-fold (from 0.1 to 0.3
mg/kg/week). This increase in dose did not effect a further decrease in
tumor size or a decrease in the growth rate of the patient’s
nonhepatic lesions. At week 93, his measurable lesions continued to
remain stable, but progression was documented outside of the liver, and
gastrointestinal bleeding felt secondary to progressive
gastrointestinal disease persisted. Treatment with Vitaxin was
discontinued at week 93.

Patient 6 with a diagnosis of metastatic breast cancer demonstrated
stable disease with the first cycle of treatment and went on to receive
three additional cycles of therapy. Although the tumor growth rate
during treatment with Vitaxin appeared to be less than that expected
(as extrapolated form her prior tumor measurements), each tumor
evaluation demonstrated slight tumor growth that eventually exceeded
25% of her baseline tumor measurements.

Patient 17, who was diagnosed with a parotid adenocarcinoma metastatic
to the lung, initially demonstrated continued progression after
his first cycle of treatment with Vitaxin. However, within 6 weeks
after treatment, in the absence of further therapy, the patient’s
tumor demonstrated a modest decrease in volume. This suggested that
Vitaxin was inducing a late response in the patient’s tumor. The
patient subsequently received a 24-week course of Vitaxin without
evidence of tumor regression.

No significant adverse events were noted in any of the three patients
undergoing continued treatment with Vitaxin.

DISCUSSION

This study reports the use of targeted antiangiogenic
therapy for cancer in patients with late stage cancer. At the doses
used, Vitaxin can be safely administered over prolonged periods without
toxicity. However, further dose escalation was limited by drug supply
and as such, no statement can be made about the toxicity of Vitaxin at
doses >4 mg/kg/week. Nonetheless, although this study did not
determine the optimum dose or schedule for Vitaxin, the plasma
concentration achieved at doses of ≥1 mg/kg/week was in excess of the
concentration required for saturation of theα
νβ3 receptors
in vitro (25 μg/ml; Ref. 15
).

Of 14 evaluable patients, 8 either demonstrated disease stabilization
or a partial response. Furthermore, in one patient, treatment resulted
in a partial tumor response that was maintained for 22 months as
determined by measurements in the indicator lesions. In a second
patient, slight tumor shrinkage was noted only after the first cycle of
therapy was completed. These findings support the notion that targeting
of the vascular ανβ3
integrin may provide clinical benefit to patients with various tumor
types without causing significant side effects.

Because of the lack of toxicity, we were unable to determine a
traditional maximally tolerated dose of Vitaxin when administered
weekly for 6 weeks. Given that this study was not designed with
adequate power to evaluate response as an end point, the single partial
response seen at 0.1 mg/kg/week does not serve to define this as the
optimal dose or schedule of Vitaxin. As with many biological therapies
in clinical trials, subsequent studies with Vitaxin will require the
establishment of a surrogate marker for clinically significant
antiangiogenic activity or will need to evaluate other measures of
patient benefit to define the optimum dose and schedule.

The pharmacokinetics of Vitaxin are similar to the pharmacokinetics
reported for other humanized monoclonal antibodies (16)
.
Vitaxin demonstrated a dose-dependent half-life that ranged from
14 h at the lowest dose evaluated to 138 h at the highest
dose. Vitaxin appeared to demonstrate a trend toward nonlinearity,
which has also been reported for other antibodies studied over a broad
dose range. No accumulation of Vitaxin was seen over the 6 weeks of
treatment. In addition, as with other humanized antibodies, Vitaxin
failed to elicit a human-anti-human antibody response in
patients.

Although significant promise exists for treatments that target tumor
vasculature, issues remain that will require resolution before such
therapy can be recommended for routine clinical use. One such issue is
the effect of antiangiogenic therapy on wound healing and mucosal
bleeding. During our study, one patient demonstrated bleeding after a
tumor biopsy at week 3. This bleeding was judged to be consistent with
biopsies of the scalp by the treating surgeon. Although this could be
interpreted as representing an increased risk for bleeding with Vitaxin
therapy, this same patient demonstrated no increased bleeding with a
cutaneous tumor resection at week 6. Subsequent patients who underwent
tumor biopsies both before and after treatment with Vitaxin
demonstrated no increase in bleeding or an inhibition of wound repair.
Although our preliminary experience suggests that treatment with
Vitaxin does not increase the risk of bleeding, we cannot rule out an
effect of Vitaxin on more significant wound healing, such as that
associated with dental procedures and/or major trauma.

A second issue involves the ability of antiangiogenic therapies to
effect a tumor response in all tumors present in a given patient. As
was observed in patient 1, even at the time of discontinuation of
therapy, several metastases remained stable, whereas other metastases
progressed. Understanding the mechanism behind this observation may
allow for the development of better therapies. One explanation of this
observation is that differing sensitivity to antiangiogenic therapies
exists between established as opposed to developing tumor vessels. An
alternative explanation is that different metastases may elaborate
different levels of angiogenic stimulation and therefore, may differ in
their sensitivity to therapy (17)
.

Given these observations, antiangiogenic therapy may prove most
useful in the treatment of patients with minimal residual disease or in
patients with occult metastatic disease. Antiangiogenic therapy may
also prove useful in combination with other classes of anticancer or
antiangiogenic drugs that operate through distinct mechanisms. For
these reasons, an ideal antiangiogenic therapy would possess minimal
toxicity and the ability to be administered over a prolonged period of
time. Vitaxin’s lack of toxicity and ease of administration should
allow for combination therapy with both chemotherapy and irradiation
therapy. Development of Vitaxin appears justified with future trials
evaluating the role of Vitaxin in longer treatment schedules and in
combination with other anticancer agents.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.